Observation of Small Cluster Formation in Concentrated Monoclonal Antibody Solutions and Its Implications to Solution Viscosity

Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals. It is hypothesized that some concentrated mAb solutions exhibit formation of a solution phase consisting of reversibly self-associated aggregates (or reversible clusters), which is speculated to be responsible for their distinct solution properties. Here, we report direct observation of reversible clusters in concentrated solutions of mAbs using neutron spin echo. Specifically, a stable mAb solution is studied across a transition from dispersed monomers in dilute solution to clustered states at more concentrated conditions, where clusters of a preferred size are observed. Once mAb clusters have formed, their size, in contrast to that observed in typical globular protein solutions, is observed to remain nearly constant over a wide range of concentrations. Our results not only conclusively establish a clear relationship between the undesirable high viscosity of some mAb solutions and the formation of reversible clusters with extended open structures, but also directly observe self-assembled mAb protein clusters of preferred small finite size similar to that in micelle formation that dominate the properties of concentrated mAb solutions.     

Efficacy of <Emphasis Type="Italic">Neoseiulus californicus</Emphasis> and <Emphasis Type="Italic">Phytoseiulus persimilis</Emphasis> in suppression of <Emphasis Type="Italic">Tetranychus urticae</Emphasis> in young clementine plants

Tetranychus urticae is one of the most damaging tetranychid mites affecting clementine orchards in Spain, where natural control is insufficient. Furthermore, in clementine nurseries, tender foliage is highly susceptible to attack and natural enemies are almost always absent. Therefore, acaricides are often used indiscriminately. Alternative control measures are necessary, both in commercial orchards and clementine nurseries. In order to assess the efficacy of inoculative releases of N. californicus and P. persimilis to reduce T. urticae populations in young Spanish clementine plants, a semi-field experiment was conducted and repeated in three seasons (spring, summer and autumn). Phytoseiulus persimilis was highly effective in reducing both T. urticae infestations and the damage level inflicted on plants at both release rates evaluated (40 and 80 phytoseiids/plant) and all three periods considered. By contrast, N. californicus demonstrated low performance under certain conditions. The results of this study could be adapted and transferred to nurseries and young citrus plantations.     

How essential is the ‘essential’ active-site lysine in dihydrodipicolinate synthase?

Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52), a validated antibiotic target, catalyses the first committed step in the lysine biosynthetic pathway: the condensation reaction between (S)-aspartate β-semialdehyde [(S)-ASA] and pyruvate via the formation of a Schiff base intermediate between pyruvate and the absolutely conserved active-site lysine. Escherichia coli DHDPS mutants K161A and K161R of the active-site lysine were characterised for the first time. Unexpectedly, the mutant enzymes were still catalytically active, albeit with a significant decrease in activity. The k_cat values for DHDPS-K161A and DHDPS-K161R were 0.06 ± 0.02 s⁻¹ and 0.16 ± 0.06 s⁻¹ respectively, compared to 45 ± 3 s⁻¹ for the wild-type enzyme. Remarkably, the K_M values for pyruvate increased by only 3-fold for DHDPS-K161A and DHDPS-K161R (0.45 ± 0.04 mM and 0.57 ± 0.06 mM, compared to 0.15 ± 0.01 mM for the wild-type DHDPS), while the K_M values for (S)-ASA remained the same for DHDPS-K161R (0.12 ± 0.01 mM) and increased by only 2-fold for DHDPS-K161A (0.23 ± 0.02 mM) and the K_i for lysine was unchanged. The X-ray crystal structures of DHDPS-K161A and DHDPS-K161R were solved at resolutions of 2.0 and 2.1 Å respectively and showed no changes in their secondary or tertiary structures when compared to the wild-type structure. The crystal structure of DHDPS-K161A with pyruvate bound at the active site was solved at a resolution of 2.3 Å and revealed a defined binding pocket for pyruvate that is thus not dependent upon lysine 161. Taken together with ITC and NMR data, it is concluded that although lysine 161 is important in the wild-type DHDPS-catalysed reaction, it is not absolutely essential for catalysis.     

Crystal structure and molecular modeling study of N-carbamoylsarcosine amidase Ta0454 from Thermoplasma acidophilum

A crystal structure of the putative N-carbamoylsarcosine amidase (CSHase) Ta0454 from Thermoplasma acidophilum was solved by single-wavelength anomalous diffraction and refined at a resolution of 2.35 Å. CSHases are involved in the degradation of creatinine. Ta0454 shares a similar fold and a highly conserved C-D-K catalytic triad (Cys123, Asp9, and Lys90) with the structures of three cysteine hydrolases (PDB codes 1NBA, 1IM5, and 2H0R). Molecular dynamics (MD) simulations of Ta0454/N-carbamoylsarcosine and Ta0454/pyrazinamide complexes were performed to determine the structural basis of the substrate binding pattern for each ligand. Based on the MD-simulated trajectories, the MM/PBSA method predicts binding free energies of ?24.5 and ?17.1 kcal/mol for the two systems, respectively. The predicted binding free energies suggest that Ta0454 is selective for N-carbamoylsarcosine over pyrazinamide, and zinc ions play an important role in the favorable substrate bound states.     

Influence of incubation conditions on the specific methanogenic activity test

The main objective of this work was to evaluate the influence of incubating conditions on the specific methanogenic activity (SMA) test in order to harmonize the test protocol. For this serum bottles were incubated with anaerobic sludge (from UASB reactor treating domestic sewage) in factorial planned experiments which assessed the influence of the temperature, substrate concentration, food/microorganism (F/M) ratio, presence of yeast extract in the medium, as well as type of carbon and nutrient solution. The results showed that the tested methane measuring methods (volumetric with biogas characterization, volumetric with gas wash in alkaline solution, and manometric by using the OxiTop^® system) presented a similar performance. The maintenance of a small gaseous phase volume (e.g. 10% of the total volume) resulted in higher SMA values; and the ideal substrate concentration for the SMA test ranged from 0.5 to 3.0 gCOD/l since higher acetate concentration caused sludge inhibition. The suggested temperature for the test is 35°C and the best F/M ratio varied from 0.125 to 0.750 gCOD/gVS, and this seemed to be the most influent parameter for the SMA test. Finally tests performed with nutrient solution complemented by yeast extract resulted in the highest SMA values.     

Asymmetric synthesis of enantiomerically and diastereoisomerically enriched 4-[F or Br]-substituted glutamic acids

A novel simple synthetic protocol for the preparation of both (2S,4R)- and (2S,4S)-FGlu, applying Michael addition of methyl α-fluoroacrylate to a Ni^II complex of glycine Schiff base with BPB, was elaborated. In addition, same reaction of mentioned complex with ethyl α-bromoacrylate leads to the Ni^II complex of the Schiff base of BPB with (2S,4R)-4-bromo-glutamic acid monoester, that can be transformed into the corresponding complexes of 1-aminocyclopropane-1,2-dicarboxylic acid. The decomposition of the diastereoisomerically pure complexes leads to corresponding enantiomerically enriched (ee > 98%) amino acids.     

Design and evaluation of 3,6-di(hetero)aryl imidazo[1,2-a]pyrazines as inhibitors of checkpoint and other kinases

A range of 3,6-di(hetero)arylimidazo[1,2-a]pyrazine ATP-competitive inhibitors of CHK1 were developed by scaffold hopping from a weakly active screening hit. Efficient synthetic routes for parallel synthesis were developed to prepare analogues with improved potency and ligand efficiency against CHK1. Kinase profiling showed that the imidazo[1,2-a]pyrazines could inhibit other kinases, including CHK2 and ABL, with equivalent or better potency depending on the pendant substitution. These 3,6-di(hetero)aryl imidazo[1,2-a]pyrazines appear to represent a general kinase inhibitor scaffold.     

Retention time prediction using the model of liquid chromatography of biomacromolecules at critical conditions in LC‐MS phosphopeptide analysis

LC combined with MS/MS analysis of complex mixtures of protein digests is a reliable and sensitive method for characterization of protein phosphorylation. Peptide retention times (RTs) measured during an LC‐MS/MS run depend on both the peptide sequence and the location of modified amino acids. These RTs can be predicted using the LC of biomacromolecules at critical conditions model (BioLCCC). Comparing the observed RTs to those obtained from the BioLCCC model can provide additional validation of MS/MS‐based peptide identifications to reduce the false discovery rate and to improve the reliability of phosphoproteome profiling. In this study, energies of interaction between phosphorylated residues and the surface of RP separation media for both “classic” alkyl C18 and polar‐embedded C18 stationary phases were experimentally determined and included in the BioLCCC model extended for phosphopeptide analysis. The RTs for phosphorylated peptides and their nonphosphorylated analogs were predicted using the extended BioLCCC model and compared with their experimental RTs. The extended model was evaluated using literary data and a complex phosphoproteome data set distributed through the Association of Biomolecular Resource Facilities Proteome Informatics Research Group 2010 study. The reported results demonstrate the capability of the extended BioLCCC model to predict RTs which may lead to improved sensitivity and reliability of LC‐MS/MS‐based phosphoproteome profiling.